Includes bibliographical references. / Nitrile hydratases can be used as industrial biocatalysts. They catalyze the conversion of nitriles to their corresponding amides. These industrial biocatalysts have recently gained attention due to the economic production of industrial commodities such as acrylamide and nicotinamide (Thomas et at., 2002). Nitrile hydratases (NHases) are metalloenzymes made up of α and β subunits, and exist mostly as heterotetramers (αβ)₂. They are categorized into Co-containing and Fe-containing types depending on their co-factor requirements.

Word processed copy. / Includes bibliographical references (leaves 82-90). / The purpose of this study was to investigate the possible prion-like properties of the N-terminal domains of the winged-helix transcription factor FoxG1.

Bibliography: leaves 180-218. / The objective of this thesis was to investigate novel and plant-based vaccines against the Human papillomavirus type 16 (HPV-16), which is primarily responsible for cervical cancer. As a first study, the L1 gene of a South Africna variant of HPV-16 (L1 504) and a mutant (504[ΔA266T]), where the alanine at 266 was mutated to a threonine, were expressed in insect cells by recombinant baculovirus, and the resulting virus-like particles (VLPs) were tested with a panel of well-characterised monoclonal antibodies (Mabs).

Includes bibliographical references (p. 160-191). / Resurrection plants are unique in that their vegetative tissue has the ability of survive reversible dehydration to an air-dry state. The widespread African resurrection plant Myrothamnus flabellifolia (Welw), woody shrub, is one of the largest of these plants. In addition to its resurrection ability it is also an important medicinal plant and is used by local tribes in the various regions where it grows to treat a wide variety of ailments. This thesis has investigated a number of morphological, ultrastructural and biochemical adaptations of the leaves of M. flabellifolia to dessication and associated stresses. The main aim of this thesis was to ascertain the role of polyphenols and the cell wall of M. flabellifolia in relation to its desiccation tolerance.

Includes bibliographical references (leaves 207-241). / The principal object of this thesis was to investigate the feasibility of developing a novel plant-derived vaccine against Human papillomavirus type 11 (HPV -11), the causal agent of genital warts and laryngeal condylomas. A secondary objective was a proof-of-concept study using Cottontail rabbit papillomavirus (CRPV), as this is a viable animal model for human papillomavirus vaccines. Accordingly, I investigated and compared the potential of two different plant expression systems.

This study describes the molecular characteristics of STEC isolated from the pastoralist community of Nyabushozi in Southwestern Uganda. Faecal samples or rectal swabs of children with diarrhoea obtained in phases 1 and 2 were investigated for the presence of STEC by PCR detection of stx genes. During phase 1, cattle reared on range which were associated with households of sick children were investigated in parallel to the children for STEC excretion. STEC was isolated from E. coli in 7 of 80 (8.8%) children and in 15 of 216 (6.9%) bovines in phase 1. Similarly, STEC was isolated from 11 of 142 (7.7%) E. coli carrying children and 3 of 45 (6.7%) water samples in phase 2. Molecular characterization further ascertained the genetic relatedness of STEC. PFGE pro les of up to 10 colonies obtained from an individual source (child, bovine or water) and in total 185 STEC colonies were analysed. Nine pro les from 43 colonies (phase 1) and 15 pro les from 38 colonies (phase 2) obtained from children were not or were distally related, indicating the genetic diversity of clinical STEC. The intra-host analysis of STEC pro les revealed that strains from 11 of the 13 children exhibited multiple clonal subgroups. The 101 colonies from 15 bovines clustered in 18 di erent pro les. Clonal subgroups were observed in multiple STEC colonies from 11 of 12 bovines. Closely related pro les indicated that STEC isolated from two children (Hh2 and Hh4) was acquired from bovines or their environment. While none of the clinical or bovine STEC were related to 5 genetically diverse water strains. A single isolate of STEC representing each PFGE pro le in association with stx gene content was serotyped for the O antigen. Twenty four bovine STEC were typed into 10 O serogroups including O8, O76, O111 and O113, which were also identi ed among the clinical STEC. The 25 clinical STEC belonged to 15 serogroups of which O29, O149 and O176 are being reported for the rst time as clinical STEC. STEC xxi Abstract O166 was isolated from a child and water during the same sampling, indicating the potential health hazard of drinking STEC-contaminated water. The production of Shiga toxin (Stx) investigated using Duopath Verotoxin detection kits showed that a majority of STEC from di erent sources produced Stx1 or Stx2 or both Stx. Using PCR or PCR-RFLP assays, stx2 and eae gene types were analysed. Variant stx2 vhc was most prevalent and closely associated with stx2d2 in clinical and bovine STEC. The frequency of eae-positive STEC among clinical and bovine STEC was 15 of 25 (60%) and 14 of 24, (58.3%), respectively. eae- 2/ was predominant among the bovine STEC, eae- / in clinical STEC, while eae- 1 was associated with STEC from di erent sources including water. Previously undescribed eae-positive serogroups O28ac, O113, O142 and O158 were identi ed. Studies of the genetic background showed that both clinical and bovine STEC obtained in phase 1 predominantly belonged to phylogenetic group A and B1, while phase 2 clinical and water STEC belonged to group D and A, respectively. Seropathotype classi cation of clinical STEC, separated most strains (20 of 24 strains) into seropathotype D. These STEC belonged to phylogenetic groups A, B1 and D. Thus, the characterised genetic attributes of STEC from Nyabushozi suggests that the pathogens have the potential to cause a wide spectrum of childhood illnesses ranging from mild to bloody diarrhoea and haemolytic uraemic syndrome. xxii

GLUT4 protein is the major glucose transporter in skeletal muscle and is vital in the maintenance of euglycemia (17; 108). Underexpression of GLUT4 or impairement of its translocation from intracellular compartments to the cell surface, are linked to diminished glucose transport, hyperglycemia and type II diabetes (59; 61; 153). Type II diabetes can be alleviated by increasing GLUT4 expression (223). Previous reports have shown that overexpression of NRF-1 and activation of CaMKII increases GLUT4 expression but the mechanisms involved have not be characterized (10; 173). Therefore, the objective of this thesis was to investigate the molecular mechanisms by which NRF-1 and CaMK II regulate GLUT4 expression in C2C12 myocytes. We engineered C2C12 cells that overexpressed NRF-1 in response to doxycycline (Dox) using a Tet-On gene expression system and assessed the effects of NRF-1 overexpression on: a) MEF2A, GLUT4 and δALAS proteins by western blot, and b) the binding of NRF-1 to mef2a and δalas genes and MEF2A to the glut4 gene, by chromatin immunoprecipitation assay (ChIP). The importance of MEF2A in NRF-1-induced increase in GLUT4 expression was investigated by silencing MEF2A expression using small interference RNA (siRNA). CaMK II was activated in wild-type C2C12 myocytes using 10 mM caffeine and was inhibited by 25 μ M KN93. Acetylation of histones in the vicinity of NRF-1 and MEF2A binding sites on the mef2a and glut4 genes, respectively, were assessed by ChIP assay. HDAC5 nuclear export was assessed by immunocytochemistry and mRNA levels by qRT-PCR. Overexpression of NRF-1 resulted in ~3-fold increases in mef2a-bound NRF-1 and glut4 -bound MEF2A at 6 h and 8 h post Dox treatment, respectively. MEF2A and GLUT4 proteins were both increased ~1.6-fold at 6 h and 18 h post Dox treatment. Silencing of MEF2A caused a marked downregulation of GLUT4 expression in NRF-1-overexpressing cells.

No description available.

No description available.

Abalone are marine gastropods that command a very high market price, particularly in the Far East where they are a highly sought after sea food delicacy. South Africa has a rapidly developing abalone aquaculture industry, based on the cultivation of Haliotis midae in landbased race-way systems. The relatively slow growth rates of abalone represent a major constraint on the abalone aquaculture industry. However, there is mounting experimental evidence showing that the health and physiology of aquacultured species can be improved through the prophylactic use of probiotic bacteria. Previous research by Macey and Coyne (2005) showed that H. midae fed a high protein artificial diet, ABFEED(R) S34, supplemented with the bacterium Vibrio midae SY9 have enhanced digestion, growth and immune responses. Probiotic microorganisms are thought to function in a variety of ways, which include the secretion of extracellular enzymes that may enhance digestion in the host organism. However, most of the investigations conducted on probiotic microorganisms for aquacultured species have failed to elucidate the exact mode of action. In this study, the predominant V. midae SY9 extracellular alkaline protease, VmproA, was investigated in an attempt to determine the role of VmproA in the growth enhancing probiotic effect observed by Macey and Coyne (2005) for abalone fed V. midae SY9 supplemented feed. The V. midae SY9 gene, vmproA, encoding the protease was cloned from a previously constructed genomic library and characterised. Nucleotide sequencing and analysis indicated that vmproA encodes a protein, VmproA, which has high similarity to a Vibrio alginolyticus extracellular detergent resistant alkaline serine protease. Furthermore, during the course of this investigation it became apparent that VmproA may represent an extracellular alkaline detergent-stable, member of the proteinase K-like subfamily of the subtilase superfamily of serine proteases. The detergent-stable protease gene, vmproA, was targeted for gene mutagenesis through vmproA gene duplication and disruption, resulting in the construction of the mutant strains V. midae SY9Pro2 and V. midae SY9Mut2, respectively. VmproA gene duplication and disruption did not significantly influence the growth of the mutant strains in batch culture in comparison to V. midae SY9. V. midae SY9Pro2 produced and secreted VmproA and had equivalent levels of extracellular protease activity to V. midae SY9 when cultivated in a high protein medium. However, insertional inactivation of vmproA resulted in a loss of VmproA production and secretion. This also resulted in a significant reduction in the extracellular protease levels produced by V. midae SY9Mut2 in comparison with that of V. midae SY9. The effect of dietary supplementation with either V. midae SY9, V. midae SY9Pro2 or V. midae SY9Mut2 on H. midae growth performance was investigated in a growth trial. The basal diet of ABFEED(R) S34 weaning chips was separately supplemented with the V. midae SY9 strains by vacuum impregnation so as to achieve a viable cell concentration of greater than 108 CFU g-1 ABFEED(R). After 180 days, H. midae receiving either the V. midae SY9Pro2 or V. midae SY9Mut2 supplemented diets displayed significantly (P<0.05) enhanced growth parameters compared to abalone fed either the basal diet or the V. midae SY9 supplemented diet. However, there was no significant difference (P>0.05) between animals fed the V. midae SY9 supplemented diet or the control diet. In situ alkaline protease levels within the crop/stomach and intestinal digestive tract regions were significantly enhanced (P<0.05) in H. midae fed V. midae SY9 supplemented ABFEED(R) S34 compared to animals fed the basal diet or the basal diet supplemented with either V. midae SY9Pro2 or V. midae SY9Mut2. In situ hybridization and immunohistochemistry were employed to examine the in vivo localisation of dietary supplemented V. midae SY9 cells and VmproA within the H. midae digestive tract. V. midae SY9 was chromosomally tagged with the mini-Tn10-gfp-kan transposon and the resulting strain, V. midae SY9